There is a growing need for new antimicrobial agents in the clinic. Antimicrobial drug resistance is rapidly spreading among pathogenic microorganisms of all types, but in sharp contrast to these emerging threats, the number of new antibiotics in the market is constantly decreasing, and the vast majority of new antibiotics represent modifications on chemical antibiotic classes that are already in clinical use. This proposal will utilize a method we recently developed for the discovery of thousands of genes toxic to bacteria, in order to identify potentially new classes of antibacterial agents. The approach is based on a by-product of the process of genome sequencing, where initial assemblies invariably contain gaps due to DNA fragments that cannot be successfully propagated in bacteria. In our pilot analysis of 79 finished microbial genomes, we discovered that many of these uncloneable gaps are caused by genes that are reproducibly toxic to E. coli, including novel antimicrobial peptides, toxic non-coding RNAs, and restriction enzymes. In this proposal we will analyze the gap content of approximately 1,500 finished microbial genomes, sampling all major phyla of the eubacterial and archaeal kingdoms, and identify a comprehensive set of an estimated ~40,000 genes toxic to E. coli. Toxic genes that are of unknown function will be evaluated in assays targeted at 1) identifying and studying antimicrobial peptides and novel antibiotic biosynthesis gene clusters 2) identifying and studying non-coding RNAs that are toxic to E. coli and 3) identifying and studying restriction enzymes that are present among the toxic genes. A public database of all genes toxic to E. coli and associated functional information from the 1,500 analyzed genomes will be developed. This database is anticipated to benefit a broad community of researchers studying antimicrobials, non-coding RNAs, restriction enzymes, and other gene functions resulting in toxicity to E. coli. PUBLIC HEALTH RELEVANCE: The rapid spread of antibiotics resistance among disease-causing bacteria forms a threat to public health. This proposal presents a new approach that facilitates the discovery of thousands of genes that are toxic to bacteria. This approach will be used to detect and develop new antimicrobial agents as well as study a new class of RNA molecules that are toxic to bacteria.